Lipid A Mimetics Based on Unnatural Disaccharide Scaffold as Potent TLR4 Agonists for Prospective Immunotherapeutics and Adjuvants

Abstract TLR4 is a key pattern recognition receptor that can sense pathogen‐ and danger‐ associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro‐inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species‐specific, and barely predictable structure‐activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan‐based lipid A‐mimicking molecules that can activate human and murine TLR4‐mediated signaling with picomolar affinity. Exploiting crystal structure ‐ based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible β(1→6)‐linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non‐reducing βGlcN(1↔1′)βGlcN scaffold. Excellent stereoselectivity in a challenging β,β‐1,1′ glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of β,β‐1,1′‐linked diglucosamine‐derived glycolipids entailing multiple long‐chain (R)‐3‐ acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D‐molecular shape and conformational rigidity of unnatural β,β‐1,1′‐linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4‐mediated signaling in a species‐independent manner.


Introduction
TLR4, an important component of the innate immune system, is a germline-encoded pattern-recognition receptor (PRR) responsible for the induction of defensive innate immune responses against bacterial infection. Activation of TLR4 by bacteriaderived pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) as well as by danger associated molecular patterns (DAMPs) which can be generated during viral or bacterial infection or by several endogenous molecules results in induction of diverse intracellular pro-inflammatory signaling pathways. Pathologic dysregulation of the TLR4mediated signaling can lead to acute or chronic inflammation and contribute to progression of a number of human diseases such as allergic asthma, atherosclerosis, arthritis, cancer and Alzheimer disease. [1] Importantly, TLR4 activation also promotes antigen processing and presentation through augmented expression of costimulatory molecules on the surface of antigen-presenting cells (APCs) which are the key parameters in initiation and regulation of the adaptive immunity. [2] Thus, TLR4 agonists represent firstÀ choice candidates for development of novel vaccine adjuvants. [3] TLR4 is broadly expressed on the cell surface of immune cells such as monocytes, macrophages, and dendritic cells where it responds to various "danger" signals such as circulating PAMPs or DAMPs, as well as in tissuerelevant cell populations, for example in the lung and bronchial epithelium, in the heart, intestinal and other tissues.
The major TLR4-activating PAMP -lipopolysaccharide (LPS, a heterogeneous glycan of ca. [10][11][12][13][14][15][16][17][18][19][20] found in the outer membrane of Gram-negative bacteria contains a relatively small glycolipid fragment (ca. 2 kDa) representing the minimal entity recognised by the TLR4 complex. [4] The LPS-recognition cascade involves several soluble and membrane-bound proteins which "deliver" LPS to the TLR4 complex where the lipid A portion of LPS can be recognized and successively bound by a secreted accessory molecule myeloid differentiation factor-2 (MD-2) that is physically associated with TLR4. [4b,5] LPS-driven dimerization of two TLR4/MD-2/LPS complexes leads to recruitment of cytosolic adaptor molecules and the assembly of intracellular multiprotein signaling platforms. [6] The latter event ultimately results in the activation of transcription factors such as NF-kB, followed by induction of expression of interferons and cytokines. [7] The LPS-sensing receptor TLR4 was highlighted as therapeutic target already two decades ago and a number of genetically engineered LPS variants, isolated or biochemically modified lipid A derivatives as well as several synthetic compounds were suggested as candidates for pharmacological manipulation of the TLR4 system. [8] However, very few TLR4 agonists were licensed for therapeutic application: MPLA (monophosphoryl lipid A-a dephosphorylated/deacylated derivative of S. minnesota lipid A) [9] and its synthetic analogue GLA are currently in use as vaccine adjuvants along with E6020 originally developed by EISAI. [3b] Especially attractive are modern developments in the syntheses of self-adjuvanting vaccine candidates where a TLRligand is covalently linked to an antigenic component. [10] Therapeutic activation of the TLR4 signaling was suggested to improve the treatment outcome of disorders with immunopathological background such as asthma, allergy, arthritis, cancer or Alzheimer disease-related pathology. [11] Despite extensive and vastly successful research, the structure-activity relationships in sensing LPS/lipid A by the TLR4/MD-2 complex are vaguely defined which reflects a very complex nature of ligand-protein interactions and a pronounced species-specificity (human versus mice) in ligand recognition by the TLR4 system. This requires the development of novel structurally defined TLR4 ligands suitable for tailored modulation of the TLR4-mediated immune responses in a welldefined, predictable, and species-independent manner.

Results and Discussion
Lipid A, the endotoxic entity of LPS, is a β(1!6)-diglucosamine -derived glycolipid that commonly contains two phosphate groups and a number of β-hydroxylated long-chain acyl tails. The phosphate groups are linked at positions 1-and/or 4'-and the β-hydroxyalkanoyl and/or β-alkanoyloxyalkanoyl chains are 2,2'-and/or 3,3'-linked ( Figure 1A). [12] The presence of both phosphate groups of lipid A is essential for proper recognition by the TLR4/MD-2 complex, [4b,13] besides, number, length and distribution of lipid chains also control the TLR4 activating potential of LPS. [8b] Along these lines, lipid A variants containing six long-chain (C 12 À C 14 ) (R)-3-hydroxyalkanoyl or/and (R)-3alkanoyloxyalkanoyl residues (such as E. coli and N. meningitidis lipid A) perform as highly endotoxic TLR4 ligands, whereas underacylated lipid A variants (penta-to tetra-lipidated) are often inactive ( Figure 1A). [14] The interior of a deep hydrophobic binding pocket of MD-2 which is lined with Leu-and Phe-residues can accommodate up to five C 12 À C 14 lipid chains of LPS, whereas the rim of the binding pocket of human MD-2 is crowned with multiple positively charged Arg and Lys residues which can establish salt bridges with the phosphate groups of lipid A. [4b,15] The binding of a typical TLR4 agonist, hexaacylated LPS of E. coli, by the TLR4/MD-2 complex induces receptor complex dimerization and the assembly of a hexameric [TLR4/MD-2/LPS] 2 complex ( Figure 1B). [4b,16] The latter occasion brings cytosolic TIR domains of TLR4 into near vicinity which directs the recruitment of adaptor proteins resulting in the assembly of a macromolecular signaling complex known as "Myddosome" which, in turn, triggers induction of the pro-inflammatory signaling cascades. [6a,17] The dimerization of the TLR4/MD-2/ligand complex is enabled through hydrophobic attraction of the 2 N-acyl chain attached at the proximal GlcN residue of lipid A towards hydrophobic side-chains of the protein complex (F126/Y131 residues of MD-2 and F440/F463 of the second TLR4*) defined as a "secondary dimerization interface" (Figure 2A ). [5b,18] Our design of lipid A mimetics was based on the analysis of co-crystal structures of the TLR4-activating lipid A variants: E. coli Ra-and Re-LPS in complex with human (h)TLR4/MD-2 complex (PDB Code: 3FXI) or mouse (m) TLR4/MD-2 complex (PDB Code: 3VQ2), respectively (Figure 2A and B). In agreement with the PDB 3FXI and 3VQ2, five lipid chains of Ra(Re)-LPS molecule are fully buried in the hydrophobic binding pocket of MD-2, whereas the sixth 2-N-lipid chain is showing to solvent ( Figure 2A). This lipid chain is involved in the formation of a secondary dimerization interface with another TLR4*/MD*-2/LPS complex which is decisive for induction of the downstream signaling cascades. [4b,19] Commonly, very small variations in the length of lipid chains (e. g., C 14 versus C 16 ) or in the pattern of acyl chains distribution Figure 2. A) Co-crystal structure of human TLR4/MD-2/Ra-LPS E. coli complex (PDB code: 3FXI), only lipid A portion of LPS is shown for clarity. The exteriorpositioned 2 N-acyl chain (colored yellow) of hTLR4/MD-2-bound lipid A is involved in formation of a secondary dimerization interface with the second hTLR4* complex. The secondary dimerization interface is maintained via hydrophobic attraction between 2 N-acyl chain and F126/Y131 side-chains of hMD-2 and F440/F463 side-chains of the second hTLR4*. [24] B) Co-crystal structure of mouse TLR4/MD-2/Re-LPS E. coli complex (PDB code: 3VQ2), only lipid A portion of LPS is shown for clarity. The 2 N-acyl chain (colored yellow) of mTLR4/MD-2-bound lipid A is involved in formation of a secondary dimerization interface with mTLR4*. The secondary dimerization interface is supported via hydrophobic forces involving 2 N-acyl chain of lipid A and F126/Y131 of mMD-2 together with F438/F461 of the second mTLR4*. C) Molecular shape of the β(1!6)-linked diglucosamine backbone of TLR4/MD-2-bound E.coli lipid A (PDB code: 3FXI); D) 3D-tertiary structure of an artificial β,β-(1 $ 1')-linked diglucosamine scaffold. The orientation of a distal pyranose ring which entails the acylation/ phosphorylation pattern of E. coli lipid A in the target structures is set similar for both snapshots (C) and (D), such that a "skewed" topology of a proximal GlcN ring becomes evident; E) Co-crystal structure-based design of disaccharide lipid A mimetics (DLAMs) as potential TLR4 agonists. The acylation and phosphorylation pattern of "distal" GlcN corresponds to E. coli or N. meningitidis LPS, whereas "proximal" GlcN entails variable acylation and phosphorylation patterns. Images were generated with PyMol. along the diglucosamine backbone of native lipid A are sufficient to "reprogram" the TLR4-mediated activity and to render a TLR4 agonist inactive. [8e,12,13] This phenomenon can be rationalized by the inherent plasticity of both binding partners: the lipid A-binding co-receptor MD-2 and its natural ligand lipid A. Depending on the presence/absence and the structure of the ligand, MD-2 can unveil multiple conformational states. [16,20] The lipid A itself is an intrinsically flexible molecule, so that the molecular shape of an easily bendable three-bond linked carbohydrate skeleton of lipid A (βGlcN(1!6)GlcN) can be easily altered [21] in a course of ligand-protein interaction (Figure 2C).
Considering all these structural peculiarities we deemed that restricting conformational flexibility of a lipid A-mimicking molecule would be advantageous for customized TLR4 complex dimerization and activation. As we have recently shown, TLR4 activation induced by lipid A mimetics based on a synthetic conformationally confined α,α-1,1'-linked disaccharide scaffold could be predictably and adjustably regulated by specific chemical modifications. [22] Along these lines, we endeavored design, synthesis and biofunctional studies of conformationally confined lipid A mimetics wherein an easily rotatable glycosidic and oxymethyl linkage in the βGlc(1!6)GlcN backbone of native lipid A is exchanged for a rigid β,β-(1 $ 1') connection ( Figure 2D). The skewed relative arrangement of two pyranose rings in the nonreducing β,β-linked disacchrides [21,23] resembles the molecular shape of the MD-2-bound β(1!6)-linked diglucosamine backbone of lipid A (as in PDB: 3FXI, 3VQ1 and 3VQ2) ( Figure 2C and D); at the same time, a specific orientation of hydroxyl-and amino groups along unnatural β,β-(1 $ 1') linked diglucosamine offers appropriate attachment sites for lipid chains and phosphate groups.
As designed, the "distal" GlcN ring reflects the configuration (β-gluco-) and the acylation/phosphorylation pattern of the "distal" β-GlcN ring of E. coli lipid A, whereas the "proximal" GlcN ring (which is supposed to face the secondary dimerization interface) entails variable acylation and phosphorylation patterns ( Figure 2E). Noteworthy, all by now synthesized lipid A mimicking compounds were based either on the native β(1! 6)-[AZ1] diglucosamine backbone or on the even more flexible skeletons where one or both GlcN residues of lipid A were exchanged for linear aglycons. [3b,c,8f,24] For the synthesis of target biomolecules possessing nonsymmetrically distributed functional groups ( Figure 2E) a convergent approach inferring first the preparation of a fully orthogonally protected β,β-1,1'-linked diglucosamine scaffold followed by regioselective acylation with optically active (R)-3acyloxyacyl long-chain fatty acids of variable chain lengths and phosphorylation was envisaged. The necessity to concurrently control the stereochemistry at two anomeric centers renders the chemical synthesis of nonreducing disaccharide a formidable challenge. Approaches to 1,1'-glycosylation commonly suffer from either lack of stereoselectivity and concomitant formation of diastereomeric by-products, or moderate yields. Whereas the synthesis of α,α-1,1'-linked and β,α-1,1'-linked disaccharides received adequate attention, the assembly of β,β-1,1'À connected nonreducing disaccharides is still an abandoned topic. [25] Very few reports describe the β,β-1,1'-glycosylation, however, these studies were majorly performed with simply protected (acetylated/benzoylated or benzylated) disaccharides. [25b,c] Our glycosylation approach towards a fully orthogonally protected βGlcN(1 $ 1')βGlcN scaffold relied on a combination of common rules for 1,2-trans glycosylation on the side of glycosyl donor (participating 2-N-protecting group) and on a scrupulously elaborated structure of a matching GlcN-derived lactol acceptor. [26] Among several temporary 2-N-protecting groups tested, only 2-azido group ensured slight conformational preference towards βÀ configured lactols. Although the anomeric ratio in the 2-N 3 protected GlcN lactols varied within α/β = 1 : 1.1 to 1 : 1.3 so that a formation of diastereomeric mixture on the side of glycosyl acceptor could be expected, several electronic effects significantly improved the steric outcome of β,β-1,1'-glycosylation. Preliminary experiments in which variably substituted GlcN lactols were subjected to TMSOTf-promoted glycosylation revealed that the electronwithdrawing protecting (or functional) groups are advantageous for enhancing the β-stereoselectivity. Also, locking the pyranose ring in a 4 C 1 conformation by using 4,6-di-OÀ cyclic protecting group proved beneficial for preponderant formation of the βÀ configured products. [27] Therefore, lactol acceptor 5 was protected with electronically disarming 2-azido-and 3-Ophosphate groups, as well as with a cyclic 4,6-di-O-DTBS protecting group. To this end, lactol acceptor 5 was prepared in four steps from the known thioglycoside 1 (Scheme 1). Deacetylation of 1 with potassium carbonate afforded triol 2 which was regioselectively converted to 4,6-O-di-tert-butylsilylene-protected thioglycoside 3 using di-tert-butylsilyl bis(trifluoromethanesulfonate (DTBS(OTf) 2 ). The remaining 3-OH group in 3 was phosphorylated using phosphoramidite approach to provide 4. N-Bromosuccinimide (NBS)-mediated hydrolysis of 4 furnished lactol glycosyl acceptor 5 (α/β = 1 : 1.1).
When planning the synthesis of DLAMs having variable acylation pattern at the distal GlcN moiety (e. g., corresponding to different natural LPS structures), it would be most straightforward to remove the 4,6-di-O-DTBS group first and to acylate 4-OH [AZ2] and 6-OH [AZ3] groups at the proximal GlcN residue next (Intermediate A), followed by attachment of acyl chains of variable length/structure at the 3'-OH and 2'-NH 2 groups of the distal GlcN moiety (Scheme 2, Route I). For the synthesis of DLAMs having C 12 À C 14 acyloxyacyl chain at position 2' of the distal GlcN residue corresponding to either E. coli or N. meningitidis acylation pattern, the intermediate B having a preinstalled 2'-N-acyloxyacyl chain and a possibility to differentiate the acylation and phosphorylation pattern at 3'-OH and at 4-OH/6-OH of the proximal GlcN ring would be of interest (Scheme 2, Route II). If the "distal" GlcN residue carries permanent acylation and phosphorylation pattern corresponding to E. coli lipid A, it would be most straightforward to remove 3'-O-TBDMS and 2'-N-Troc groups followed by introduction of 2'-, 3'-acyloxyacyl lipid chains first (as in the intermediate C) and then to manipulate acylation/phosphorylation pattern at positions 4-and 6-of the proximal GlcN residue (Scheme 2, Route III).
Following the first option (Route I via intermediate A), the 4,6-di-O-DTBS group in 8 was regioselectively cleaved without affecting 3'-O-TBDMS group by application of a lowÀ concentrated solution of HF · Py to give diol 9 with nearly 90 % yield (Scheme 3).
This transformation was thoroughly optimized to reach excellent regioselectivity: multiple experiments were carried out to demonstrate that the absolute concentration (but not the number of equiv.) of fluoride reagent in reaction solution was decisive for the regioselective cleavage of 4,6-di-O-DTBS group without affecting 3-O-TBDMS protection.
The 2'-N-Troc protecting group was removed under reductive conditions (Zn in acetic acid) which liberated 2'-amino group for subsequent acylation with a branched long-chain fatty acid FA5. The attempts to introduce (R)-3-(dodecanoyloxy)tetradecanoyl residue using standard coupling reagents such as DIC or N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) brought about only sluggish and incomplete reaction and resulted in formation of multiple byproducts. Application of uronium-type coupling reagent O-(7azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) in the presence of DIPEA provided substantial improvement and the hexaacylated product 15 could be isolated in pure form, although with 42 % yield only. Sluggish reaction rate and inefficient transformation were rationalised by amphiphilicity of the tetraacylated free amine intermediate and its tendency to form micelles even in organic solutions which rendered the 2'-amino group poorly accessible for acylation reagents. Global deprotection was performed via hydrogenolysis on Pd black and the target hexaacylated bisphosphorylated ββ-DLAM909 was purified by size-exclusion chromatography on Bio-Beads SÀ X1 support using toluene-DCM-methanol as eluent.
Given unsatisfactory yields in the step of reduction of 2'-N-Troc group and the following N-acylation which were performed at the stage of a fully assembled protected glycolipid (14!15), we re-designed our synthetic route and envisaged the reductive cleavage of the 2'-N-Troc group and subsequent acylation at the very beginning of the synthesis (Intermediate B, Scheme 2). To this end, 2'-N-Troc group in 8 was removed using Zn in acetic acid at 0°C and the intermediate amine 16 was carefully purified by column chromatography which allowed for substantial improvements in the next step acylation reaction compared to application of a crude product 16 (Scheme 4). Gradual addition of activated long-chain alkanoyloxyalkanoic acid to a solution of free amine 16 also proved advantageous and resulted in formation of 17 with 74 % yield. Next, the 4,6-di-O-DTBS protecting group in 17 was regioselectively cleaved without affecting 3-O-TBDMS protection using diluted solution of HF · Py to furnish diol 18 which was subsequently doubleacylated by (R)-3-(benzyloxy)decanoic acid FA1 with DIC/DMAP as coupling reagent to give 19. The 3'-O-TBDMS group was removed by treatment with Et 3 N · 3HF to give 20, the liberated 3'-OH group was acylated by reaction with (R)-3-(tetradecanoyloxy)tetradecanoic acid FA6 activated by DIC and catalytic amount of DMAP which gave hexaacylated intermediate 21. The 4',6'-O-benzylidene acetal in 21 was regioselectively reductively opened by reaction with TfOH/Et 3 SiH in DCM at À 78°C and the liberated 4'-OH group in 22 was phosphorylated using phosphoramidite procedure to give 23. Global deprotection by hydrogenolysis on Pd black afforded ββ-DLAM919, a shorter-chain analogue of ββ-DLAM909.
To create a broader scope of lipid A mimetics for studying structure-activity relationships in ligands recognition by the TLR4 complex we aimed to explore the biological activity of ββ-DLAMs carrying acylation pattern of N. meningitidis lipid A (three long-chain lipid residues attached at each GlcN ring). To this end, we performed the synthesis of hexaacylated diphosphate ββ-DLAM933 starting from diol 18. For regioselective 6-OH-acylation, the primary 6-OH and secondary 4-OH groups in 18 had to be differentiated using protective group manipulation strategy. To avoid three additional synthetic steps, we initially explored the possibility of a direct regioselective 6-Oacylation of the 4,6-diol 18. We reasoned that the 4-OH group in 18 is heavily disarmed due to the presence of two electronwithdrawing groups (2-acetamide and 3-O-phosphate) and, therefore, will be resistant to acylation by a bulky branched long-chain fatty acid under standard acylation conditions. Indeed, upon reaction of 18 with (R)-3-(dodecanoyloxy)tetradecanoic acid FA5 in the presence of DIC/ DMAP the primary 6-OH group was acylated exclusively to give 24 with 75 % yield.
The cleavage of 3'-O-TBDMS protecting group generated a 3,3'-diol which was double-acylated with benzyl-protected βhydroxy fatty acid FA2 to give 25. Reductive opening of benzylidene acetal in 25 followed by phosphorylation of the liberated 4'-OH group using phosphoramidite procedure gave rise to a fully protected hexaacylated diphosphate 27. The cleavage of all benzyl protecting groups in the molecule by hydrogenolysis over Pd black gave rise to ββ-DLAM933 having N. meningitidis-like acylation pattern.
Immunobiological screening of three ββ-DLAMs ββ-DLAM909, 919 and 933 in TLR4/MD-2/CD14-transfected human embryonic kidney 293 cells (HEK293) revealed very low or no TLR4-activating potential ( Figure 3A). Although the acylation pattern of all three glycolipids was predicted to be highly suitable for the generation of TLR4 agonist lipid A mimetics, the 3-O-phosphate group at the proximal GlcN ring (presumable dimerization interface) was apparently misplaced and did not support appropriate ionic interactions with the second TLR4* complex. Thus, initially synthesised ββ-DLAM909, 919 and 933 failed to induce efficient dimerization of the TLR4/MD-2/ligand complexes and the TLR4-mediated signaling. We also examined whether these DLAMs could act as antagonists and inhibit the induction of pro-inflammatory signaling induced by increasing doses of E. coli LPS in TLR4/MD-2/CD14-transfected HEK293 cells ( Figure 3B). Only ββ-DLAM909 could inhibit the LPS-induced release of IL-8 (LPS concentration 100 ng/mL to 10 μg/mL), however, at higher LPS concentration (10 μg/mL) the inhibitory effect was not significant (50 %).
Based on the analysis of co-crystal structures of natural TLR4 agonists E. coli Ra-LPS in complex with hTLR4/MD-2 (PDB code: 3FXI) and Re-LPS in complex with mTLR4/MD-2 (PDB code: 3VQ2), the distance between 4'-and 1-phosphate groups of lipid A is insignificantly longer compared to the distance between 4'-and 3-phosphate groups in ββ-DLAM909/919/933 which obviously thwarts appropriate ligand-protein interaction and hampers dimerization. Consequently, to render the ββ-DLAMs biologically active, the phosphate group must be either shifted to position 4 of the proximal GlcN moiety (3!4) or, alternatively, an additional phosphate group at position 4 could be attached. Since the binding pocket of hMD-2 is crowned with several positively charged Lys and Arg side-chains which can establish ionic bridges with the lipid A phosphates (this effect was demonstrated both for agonist (PDB code: 3FXI) [4b,15] and antagonist co-crystal structures (PDB code: 2E59 and 2Z65)), [14] we hypothesised that retaining the 3-O-phosphate group at the proximal GlcN moiety could be beneficial for enhancing affinity to hMD-2. Thus, we reasoned that attachment of an additional phosphate group in position 4 would help in establishing of a proper secondary dimerization interface with the second TLR4* by virtue of ionic interactions and would support the ligand-induced receptor complex dimeriza- tion, whereas preserving the phosphate group at position 3 would tighten the binding of ββ-DLAM to hMD-2.
Simultaneously, the acylation pattern of the ββ-DLAM molecule was adjusted: since O-4 at the proximal GlcN residue was now occupied with a phosphate group, two lipid chains had to be attached at O-6 in a form of (R)-3-(alkanoyloxy)alkanoyl chain as in ββ-DLAM937 (Scheme 4, Route C). Such acylation pattern was deemed advantageous for high-affinity binding to MD-2 and further dimerization. According to our model, the shape of hydrophobic clusters formed by the long-chain lipid tails in ββ-DLAM919 and 933 could be unfavourable for binding to MD-2 and further dimerization ( Figure 3C), whereas the predicted hydrophobic profile of ββ-DLAM937 with four tightly packed lipid chains attached at the distal GlcN and one alkanoyloxyalkanoyl residue linked at position 6 of the proximal GlcN should be sufficient for both high-affinity binding to MD-2 and presentation of 6-O-acyloxyacyl chain at the secondary dimerization interface. The latter should support the assembly of a hexameric activated receptor complex [TLR4/MD-2/ββ-DLAM] 2 .
Along these lines, the 4-OH group in 24 was phosphorylated according to phosphoramidite procedure to give 28, the 3-O-TBDMS group was smoothly removed using solution of TREAT-HF in DCM followed by O-acylation with (R)-3-(tetradecanoyloxy)tetradecanoic acid FA6 (Scheme 4, Route C) Subsequent regioselective reductive opening of benzylidene acetal to form 6-O-Bn protected 31, followed by phosphorylation of the liberated 4'-hydroxyl group gave rise to a fully protected hexaacylated triphosphate 32. Global deprotection by hydrogenolysis on Pd black and purification via sizeexclusion chromatography on Bio-Beads SÀ X1 provided ββ-DLAM937.
The length and configuration of lipid chains attached at the sugar moiety exposed at the secondary dimerization interface is known to exert profound effect on the tightness of TLR4/MD-2/ ligand complex dimerization and the ensuing downstream signaling. [22a,30] For this reason, the synthesis of a short-chain analogue of ββ-DLAM937 starting from a key intermediate 9 was undertaken. To establish a more expedient synthetic route and to reduce the number of parallel steps towards a library of differently acylated DLAMs, we redesigned our initial synthetic approach by discriminating the 4-and 6-OH groups in 9 using orthogonal protective group manipulation strategy (Scheme 2, Route III). To differentiate the 4-and 6-OH groups in 9, we applied our previously developed procedure for regioselective protection of primary 6-OH group as allyloxycarbonate (Alloc) by a mild base-catalysed reaction with AllocCl. [31] Accordingly, the 6-hydroxyl group in diol 9 was reacted with allyloxycarbonyl chloride in the presence of symÀ collidine which afforded 33 as a single product in 85 % yield. The 4-hydroxyl group in 33 was acylated with levulinic acid using EDC · HCl and DMAP which provided fully orthogonally protected disaccharide 34. To reduce the number of parallel steps, the distal GlcN moiety in 34 had to be fully acylated prior to instalment of functional groups at the proximal GlcN unit (Intermediate C, Scheme 2). Thus, two branched lipid chains of different lengths (corresponding to acylation pattern of E. coli lipid A) were initially attached at position 2'-, 3'-of the distal GlcN to produce a key intermediate 37 ready for differentiation by functional groups at the proximal GlcN moiety (Scheme 5).
To this end, desilylation of 34 furnished 35 which was subsequently acylated by reaction with (R)-3-(tetradecanoyloxy)tetradecanoic acid FA6 using EDC · HCl/DMAP as a coupling reagent to give 36 (Scheme 5). Removal of the N-Troc group in 36 was accomplished under reductive conditions with zincÀ copper couple in acetic acid and the resulting amine was acylated with branched long-chain fatty acid FA5 using EDC · HCl as coupling reagent to give 37. Fully protected tetraacylated intermediate 37 was treated with hydrazine hydrate to remove the levulinoyl ester from position 4 which gave 38 in 84 % yield. Next, the liberated 4-OH group in 38 was phosphitylated with bis(benzyloxy)(diisopropylamino)phosphine in presence of 1Htetrazole, followed by oxidation with tert-butyl hydroperoxide. The latter oxidant was preferred to a standard oxidative reagent mCPBA to avoid a concomitant epoxidation of the Alloc group. Removal of the primary Alloc protecting group from 39 was accomplished by treatment with bis(triphenylphosphine)palladium chloride and tributyltin hydride in the presence of water [32] which gave 40. In the next step, the primary 6-OH group in 40 was acylated with branched long-chain fatty acid FA6 under standard conditions (EDC · HCl/ DMAP) which resulted in formation of a target 41 along with elimination by-product (due to elimination of a secondary acyl chain which gave rise to 6-O-tetradecanoyl derivative) in nearly equal amounts (1 : 1).
Elimination of a secondary acyl chain in 3-alkanoyloxyalkanoyl moieties during DMAPÀ catalysed carbodiimide-mediated coupling reactions has been described previously by us and others, [8h,24b] although the formation of elimination byproducts was reported for the acylation of unreactive and/or sterically hindered secondary, but not primary OH groups. Obviously, the electron-withdrawing effect of the 2-NHAc and 3-O-, 4-O-phosphate groups deactivated the primary 6-OH group in 40 which resulted in inefficient acylation. To find a solution for a straightforward 6-O-acylation we screened numerous protocols and applied different coupling reagents (including 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU) in presence of DBU, [33] DIC/DMAP, etc.) but all tested conditions led to formation of a hardly separable co-migrating elimination by-product which lowered the isolated yield of target 41 to 30 %.
Finally, we took advantage of the Mitsunobu reaction conditions (PPh 3 , DIAD) to acylate the primary 6-OH group in 40 which resulted in formation of 41 as a single product (Scheme 5). Next, the benzylidene acetal in 41 was regioselectively reductively opened using trifluoromethane-sulfonic acid and triethylsilane in DCM which afforded 6-O-Bn protected 42. The liberated 4-OH group was phosphitylated using bis(benzyloxy)(diisopropylamino)phosphine in the presence of 1H-tetrazole and subsequently in situ oxidized with mÀ CPBA to furnish 43. Global deprotection by hydrogenolysis on Pd black followed by purification using size-exclusion chromatography on Bio-Beads SÀ X1 gave ββ-DLAM3370, a shorter-chain analogue of ββ-DLAM937, in 85 % yield.
Next, we evaluated the ability of ββ-DLAMs to induce cytokine release in primary human immune cells by challenging human mononuclear cells (hMNC) with increasing concentrations of ββ-DLAMs and compared these responses to E. coli LPS and a potent TLR4 agonist lipid A mimetic αα-DLAM 5 [30] ( Figure 4A and B). The release of IL-6 elicited by picomolar concentrations of ββ-DLAM3370 was two-fold higher compared to ββ-DLAM937 (EC 50 = 0.8 nM and EC 50 = 1.6 nM, respectively), although both ββ-DLAMs were significantly less efficient in inducing the expression of IL-6 than powerful TLR4 agonist αα-DLAM 5 (EC 50 = 0.06 nM) ( Figure 4A). Thus, the TLR4-activating capacity of ββ-DLAMs can be readily adjusted by shortening or lengthening (for 2xCH 2 atoms) the secondary lipid chain attached at the "proximal" GlcN unit which is supposedly exposed at the secondary dimerization interface in the hexameric [TLR4/MD-2/ββ-DLAM] 2 complex.
Importantly, ββ-DLAM937 and ββ-DLAM3370 induced the expression of IL-6 in human MNC to a 100-fold lesser extent compared to E. coli LPS (EC 50 = 0.004 nM, calculated based on an average MW of 10 kDa) which reinforces the rationale of our crystal-structure-based design in an effort to achieve controlled TLR4 activation upon minimizing the LPS-related toxic effects. Regarding the release of IL-1β in hMNC, the relative efficacy (or maximum response) was higher for ββ-DLAM3370 compared to ββ-DLAM937, although the potency of response was similar (EC 50 = 2.3 vs. 2.9 nmol/mL) (Figure 4 B). Both ββ-DLAMs were 30-fold less efficient in eliciting IL-1β compared to α,α-DLAM5 [30] (EC 50 = 0.08 nmol/mL) and 100-fold less potent com-pared to E. coli LPS (EC 50 = 0.026 nmol/mL). Thus, both compounds under investigation induced the expression of IL-6/IL-1β in human primary immune cells in pico-to nanomolar concentration range and, as intended, were less potent (and less endotoxic) compared to E. coli LPS.
Since ββ-DLAM909, 919 and 933 failed to activate TLR4transfected HEK293 cells and to induce the release of cytokines in MNC (data not shown), we screened these compounds for potential ability to prevent LPS-induced TLR4 activation in human MNC. The cell culture was preincubated with the respective ββ-DLAM (10 μg/mL) or with the potent TLR4 antagonist DA193 [28,34] for 1 h and then challenged with increasing concentrations of E. coli LPS (10 to 10.000 pg/mL). In line with the data obtained in HEK293/TLR4 cells ( Figure 3B), only ββ-DLAM909 showed weak antagonist capacity in human MNC in regard to suppression of the LPS-induced release of IL-6/IL-1β ( Figure 4C and D), whereas ββ-DLAM919 and ββ-DLAM933 were inactive.
We also examined the cytokine-inducing ability of ββ-DLAMs in the human bronchoepithelial cell line Calu-3. Generally, the sensitivity of human airway epithelial cells to LPS is diminished due to insufficient expression of MD-2 [35] as well as low expression levels of the membrane-bound CD-14. [36] Accordingly, the induction of cytokine release (IL-6/IL-8) by ββ-DLAMs in bronchoepithelial cell line required higher (nanomolar) concentrations of synthetic TLR4 ligands ( Figure 5A and B). The secretion of IL-8 in Calu-3 cells was influenced by the length of secondary lipid chain at the proximal GlcN unit and  [34] D) Inhibition of LPS-induced expression of IL-1β by selected ββ-DLAMs in hMNC compared to TLR4 antagonist DA193. [34] . was correspondingly higher for the longer-chain ββ-DLAM937 at concentrations below 100 ng/mL which related to the lowlevel expression of CD-14. Both ββ-DLAMs were less potent though compared to a "reference molecule"-a strong TLR4 agonist α,α-DLAM 5. [30] To address possible species-specificity in the cellular activation by ββ-DLAMs, we analyzed the expression of TNF-α in bone marrow-derived mouse wt macrophage cell line challenged with increasing concentrations of ββ-DLAMs (Figure 5). The first series of ββ-DLAMs (ββ-DLAM909, 919 and 933) which could not activate hTLR4 and failed to induce cytokine release in human immune and epithelial cell lines was similarly inactive in murine macrophages (data not shown). In contrast, ββ-DLAM937 and ββ-DLAM3370 efficiently induced the expression of TNF-α in murine macrophages with picomolar affinity ( Figure 5C). ββ-DLAM3370 having shorter secondary lipid chain was more efficient in eliciting TNF-α in murine macrophages (EC 50 = 0.029 nmol/mL) compared to the longerchain-lipidated ββ-DLAM937 (EC 50 = 0.04 nmol/mL), whereas the response to α,α-DLAM5 was at least 2-fold stronger upregulated (EC 50 = 0.016 nmol/mL).
To assess the potential of ββ-DLAMs to activate professional antigen-presenting cells, dendritic cells (DCs) were challenged with increasing concentrations of DLAMs and the expression of pro-inflammatory cytokines IL-6 and IL-12, as well as the production of major immunosuppressive cytokine IL-10 was analysed ( Figure 6). Accordingly, immature human monocytederived DCs were stimulated with TLR4-activating ββ-DLAM937 and ββ-DLAM3370 at concentration ranging from 0.01 mM to 1000 mM or with E. coli LPS as control.
Both DLAMs induced dose-dependent release of IL-6, IL-10 and IL-12 in hDCs and the responses were generally lower than those induced by E. coli LPS ( Figure 6). The production of proinflammatory cytokine IL-6 was induced by ca. 10 nM concentration of ββ-DLAMs whereas already 0.01 nM LPS was sufficient to reach the same level of induction which highlights the DLAMs as safe, less toxic TLR4 modulators compared to LPS ( Figure 6A). ββ-DLAMs also efficiently induced the expression of immunoregulatory cytokine IL-10, a pleiotropic cytokine with crucial role in limiting the extent of inflammation and maintaining the homeostatic state ( Figure 6B). We also demonstrate that both ββ-DLAM937 and ββ-DLAM3370 could induce the release of IL-12, a critical factor for biasing the immune response towards a T helper 1 (Th1) cytokine profile, and that nano-molar doses of ββ-DLAMs were sufficient for this activity ( Figure 6C).

Conclusion
We developed a divergent synthetic strategy towards a series of lipid A mimicking biomolecules derived from an artificial β,β-1,1'-linked diglucosamine scaffold and prepared five complex glycolipids having variable acylation and phosphorylation patterns (disaccharide lipid A mimetics, ββ-DLAMs). Excellent stereoselectivity in a demanding β,β-1,1'-glycosylation which was achieved through application of torsional-and electronically-disarming orthogonal protecting groups allowed for highyielding, reproducible and upscalable (up to 1 g of glycosyl donor) glycosylation reaction. Compared to native TLR4 agonist lipid A which entails a readily bendable three-bond linked diglucosamine backbone [βGlcN(1!6)GlcN], the ββ-DLAM molecules are built on the basis of a conformationally constrained two-bond linked diglucosamine scaffold [βGlcN(1 $ 1)βGlcN] which reveals specific tertiary structure with two pyranose rings in a staggered arrangement. The particular 3D-molecular shape of DLAMs based thereof was decisive for efficient crosslinking of two TLR4/MD-2/DLAM complexes and resulting induction of the intracellular signaling. As judged by production of cytokines induced by pico-to nanomolar concentrations of ββ-DLAMs in human and murine immune cells, these synthetic glycolipids comprise a new class of unprecedentedly efficient TLR4dependent modulators of innate immune responses. The induction of somewhat lower levels of pro-inflammatory cytokines TNF-α, IL-6, IL-8 and IL-1β by ββ-DLAMs compared to LPS highlights ββ-DLAMs as safe TLR4 agonists with minimized toxicity. The capacity of novel glycolipids to elicit the release of several important cytokines which provide a critical bridge between the innate and adaptive immunity as well as the possibility to modulate cellular responses by modification of the primary chemical structure renders ββ-DLAMs eligible for future development as promising vaccine adjuvants and immunotherapeutics.

General synthetic methods
Reagents and solvents were purchased from commercial suppliers and used without further purification unless otherwise stated. Toluene was dried by distillation first over phosphorus pentoxide, then over calcium hydride and was then stored over activated 4 Å molecular sieves (MS). Solvents were dried by storage over activated MS for at least 24 h prior to use (dichloromethane 4 Å, acetonitrile and DMF 3 Å). Residual moisture was determined by coloumbometric titration on a Mitsubishi CA21 Karl Fischer apparatus and did not exceed 20 ppm. Reactions were monitored by TLC performed on silica gel 60 F254 HPTLC precoated glass plates with a 25 mm concentration zone (Merck). Spots were visualized by dipping into a sulfuric acid-p-anisaldehyde solution and subsequent charring at 250°C. Solvents were removed under reduced pressure at � 40°C. Preparative HPLC was performed on a YMC Pack SIL-06 250 × 20 mm, S-5 μm, 6 nm column or on a YMC Pack SIL-06 250 × 10 mm, S-5 μm, 6 nm column. Preparative MPLC and column chromatography were performed using silica gel 60 (0.040-0.063 mm). Size exclusion chromatography was performed using Bio-Beads SÀ X1 support (BioRad). NMR spectra were recorded on a Bruker Avance III 600 spectrometer ( 1 H at 600.22 MHz; 13 C at 150.93 MHz; 31 P at 242.97 MHz) using standard Bruker NMR software. Chemical shifts are reported in ppm, where 1 H NMR spectra recorded from samples in CDCl 3 were referenced to internal TMS and 13 C spectra were referenced to the corresponding solvent signal (77.16 ppm for CDCl 3 ). NMR spectra recorded from samples in other solvents were referenced to residual solvent signals (for CD 3 OD 3.31 and 49.00 ppm; for CD 2 Cl 2 5.32 and 53.84 ppm; for DMSO-d 6 2.50 and 39.52 ppm; for 1 H and 13 C NMR, respectively). NMR spectra recorded in CDCl 3 -MeOD (4 : 1, v/v) were referenced to residual solvent signals of CDCl 3 (7.26 ppm and 77.16 ppm; 1 H and 13 C NMR, respectively). NMR spectra recorded in CDCl 3 : MeOD (1 : 1 to 4 : 1, v/v) were referenced to residual solvent signals of MeOD (3.31 and 49.00 ppm, 1 H and 13 C NMR, respectively). 31 P NMR spectra were referenced according to IUPAC recommendations from a referenced 1 H NMR spectrum. In all 1,1'-disaccharides the NMR signals of the "distal" GlcN ring are indicated by primes. Highresolution mass spectrometry (HRMS) was carried out on acetonitrile or DCM solutions via LC-TOF MS (Agilent 1200SL HPLC and Agilent 6210 ESI-TOF, Agilent Technologies). Datasets were analyzed using Agilent Mass Hunter Software. MALDI-TOF MS was performed in negative-ion mode using a Bruker Autoflex Speed instrument with 6-aza-2-thiothymine (ATT) as matrix and ammonium sulfate as additive. Optical rotation was measured on a PerkinElmer 243B polarimeter equipped with a Haake water circulation bath and a Haake D1 immersion circulator for temperature control or an Anton Paar MCP 100 polarimeter featuring integrated peltier temperature control. All [α] D 20 values are reported in units of deg dm À 1 cm 3 g À 1 , the corresponding concentrations are reported in g/100 mL.